Based on the pCAGGS-S1254 plasmid, we generated the S-D614G variant construct by PCR-based direct mutagenesis using a pair of corresponding primers listed in materials and methods. For the product S and S-D614G, we found out that their optimal sizes should be ~7500bp. However, problems arose as there was no band at or around ~7500bp. We realized that the template DNA might be too abundant and anneal temperature might be too low (56℃) to yield a band at ~7500 bp. When we lessened template DNA and increased the anneal temperature into 58 ℃, we finally got the right size for our PCR products.
We cloned the full length S gene S-FL into a pCAGGS vector and generated a wild type S pseudovirus (pCAGGS-S). We evaluated the efficacy of virus packaging and production of the S-FL (full length). We found that the efficacy of virus packaging and production was very low. Seeing as this was not ideal for our experimentations, we tried to find a way that would be more effective. By engineering a deletion mutation, S1254 with a c-terminal 19 amino acid deletion (from 1255-1273), we discovered that the S1254 had a much higher packaging efficacy titer (3.3E+04 in S-FL, 2.7 E+05 in S1254).
b. Comparison of the pseudovirus production titers of S-FL and S1254 (1-1254)
When other teams use our parts, they can use these strategies to optimize their project. Through our trouble-shooting, when future teams are not able to create a band with the necessary base pairs, they too can alter the DNA strand length and anneal temperature. Furthermore, if teams are faced with a low effectiveness in their virus packaging and production, by inducing a mutation, they can create a method that is more efficient.